This invention relates to a plasminogen proactivator and activator which can be isolated from mammalian and avian plasma, concentrated plasma protein fractions (such as Cohn fractions) or from corresponding cell culture fluids; to methods for the isolation and purification thereof; to pharmaceutical and diagnostic methods and compositions for the use thereof; to the production of antisera thereto; and to the use of such antisera in purification of the plasminogen proactivator and activator and in measurement of plasminogen activator levels in the blood and body fluids.
As thrombin, which converts the soluble fibrinogen of the blood into insoluble fibrin, exists in the form of a proenzyme, i.e. prothrombin, so also does plasmin, the proteolytic enzyme which lyses blood clots, exist in the form of a proenzyme, i.e. plasminogen. Fibrinolysis is generally agreed to be a normal process, potentially active at all times to insure the fluidity of blood and the patency of the vasculature. Because the formation of plasmin from plasminogen is an ongoing process in the body, the clinical determination of the fibrinolytic potential (e.g., as measured by plasminogen levels in blood or other body fluids) in a given individual is of great importance in the anticipation or treatment of systemic hyperfibrinolysis; post-surgical complications; neoplastic diseases such as carcinoma of the prostrate, lung, stomach and cervix; hemorrhagic states accompanying trauma; shock; liver disease and hematologic disorders; and thromboembolic phenomena in diabetes, atherosclerosis, venous stasis and thrombophlebitis.
Plasminogen can be activated in various ways to yield the active proteolytic enzyme, plasmin. For example, fibrinokinase activity has been found in many tissues and in the plasma and urokinase is obtained from the urine, while staphylokinase and streptokinase are obtainable from bacteria; all of these enzymes can activate plasminogen to form plasmin. Of these, urokinase is most widely employed in clinical practice today, in spite of its many inherent deficiencies. These are largely due to the very short (3-15 minutes) useful half-life of urokinase following its injection into humans, as has been reported by Fletcher et al. in J. Lab. Clin. Med. 65: 713 (1965). This short half-life is due to a number of factors. For example, the molecular weight of urokinase is below the threshold for kidney retention so that a substantial fraction of every administered does is therefore repidly lost by excretion into the urine. Furthermore, urokinase exists entirely in a form which is enzymatically fully active before injection; immediately upon injection, it reacts with the protease inhibitors that are normally present in relatively high concentrations in plasma and body fluids, and is thereby inactivated. The short in vivo half-life of urokinase requires frequent injections to achieve the desired therapeutic effect; this, together with the low concentrations of urokinase present in the urine and cell culture fluids that are the sources for preparing this enzyme, makes urokinase therapy very costly. Because of the widespread interest in plasminogen proactivator and activators, many attempts have been made to obtain plasminogen activators which do not share the above deficiencies of urokinase.
The use of streptokinase as a substitute for urokinase in such therapy is limited by the fact of its bacterial origin. Since streptokinase is a protein foreign to man, injection of streptokinase gives rise to the production of neutralizing antibodies which block its action and to allergic reactions that are harmful, and potentially fatal, to the recipient. Summaria et al., in J. Biol. Chem. 251 (18): 5810-5813 (Sept. 25, 1976) describe the isolation of a human plasmin-derived, functionally active human plasmin light (B) chain derivative which, mixed in equimolar amounts with streptokinase, develops both human and bovine plasminogen activator activities. However, this derivative is reported to possess only approximately 3% of the proteolytic activity of the original enzyme.
A number of earlier workers have described other combinations of plasminogen with streptokinase, resulting in an agent which possesses thrombolytic activities similar to streptokinase without reacting against antibodies thereto; for example, see Holleman et al., U.S. Pat. No. 3,865,692.
In another approach to overcoming the inherent deficiencies of streptokinase and urokinase as plasminogen activators, other investigators have turned their attention to obtaining plasminogen activators from different materials. For example, Wagner, U.S. Pat. No. 3,555,000, describes a plasminogen activator which can be isolated from the stromata of human erythrocytes and can be electrophoretically separated from urokinase. The activator is a water-soluble protein having a molecular weight of about 20,000-50,000 and which is itself activated by small amounts of urokinase.
Hull et al., U.S. Pat. No. 3,904,480, describe the culturing of another plasminogen activator from mammalian tissue culture cells, e.g. PK-15, LLC-MK.sub.2, etc. This plasminogen activator is a water-soluble polypeptide having a molecular weight of approximately 30,000 and an activity, observed by the fibrin plate method and related to the optical density (OD) in the ultraviolet spectrum at 280 nm, of about 15,000-20,000 CTA units.
D'Hinterland et al., U.S. Pat. No. 3,998,947, describe a method for obtaining a plasminogen activator by acetone extraction of organ tissues, using a process similar to one which has long been employed in the preparation of organ tissue powders. The activator is endocellular and characterized as a glycoprotein having a molecular weight of approximately 40,000, containing from 300 to 500 CTA units per milligram and lacking esterase activity with respect to most, but not all, amino acid esters. The activator is medically useful in the treatment of arterial and venous thrombosis, and in the treatment of fibrinous deposits.
In spite of all the attempts at isolating new plasminogen activators, those heretofore employed in the prior art suffer from one or more deficiencies of limited purity and poorly reproducible potency; an extremely short half-life, thereby necessitating pharmaceutical administration by continuous slow injection, perfusion or the like, or frequent injections several times a day; susceptibility to inactivation by a number of chemicals commonly encountered in biological fluids; and induction of undesirable immunological reactions. In addition, the plasminogen activators that are prepared using tissues, extracted tissues or tissue powders as a starting material suffer from the disadvantage that they are present in very low concentrations and are accordingly difficult to purify and to obtain in reproducibly potent preparations. None of these deficiencies presently appears to apply to the newly isolated plasminogen proactivator and activator of the present invention.